Temperature Control Of Enrichment And Separation Columns In Chromatography

ABSTRACT

A method of analyzing samples includes loading a sufficient quantity of the sample onto a trap column to overload the trap column; heating an analytical column and the trap column to a greater temperature than the analytical column; and pumping a solvent, to the trap column, having a solvent composition profile that, in cooperation with a temperature differential, causes at least some of the components to elute sequentially from the trap column to the analytical column and focus on the analytical column prior to eluting from the analytical column; or optionally: loading a small-molecule sample onto a cooled portion of an analytical column; heating the analytical column; and pumping a solvent, to the heated analytical column, to elute the components from the analytical column. Chromatographic separation includes: a trap column; a separation column; a trap-column heater; a separation-column heater; a solvent pump unit; and a control unit can be used.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Nos.61/182,268 and 61/182,498, both filed May 29, 2009. The entire contentsof these applications are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to chromatography. More specifically,preferred embodiments of the invention relate to apparatus and methodsfor liquid-chromatography of complex protein-related samples and ofsmall-molecule samples.

BACKGROUND

High-performance liquid chromatography (HPLC) instruments are analyticaltools for separating, identifying, and quantifying compounds.Traditional HPLC instruments use analytical columns constructed fromstainless-steel tubing. Typically, the tubing has an inner bore diameterof 4.7 mm, and its length ranges from about 5 cm to about 25 cm.

In addition, the analytical column of an HPLC instrument typically has afritted end fitting attached to a piece of tubing. Particles, typicallysilica-based, functionalized with a variety of functional moieties, packthe tube.

To achieve optimal separation efficiency, using the completed column, anappropriate flow rate of a mobile phase is important. For a 4.7 mmdiameter column packed with 5 μm diameter particles, a desirable flowrate is typically between about 1 mL/min and about 2 mL/min. Minimizingthe presence of unswept dead volume in the plumbing of the HPLCinstrument is desirable for maintaining separation efficiency.

In an HPLC instrument, an injector is typically used to inject a sampleinto a flowing mobile phase as a discrete fluidic plug. Dispersion of aplug band as it travels to and/or from the column reduces the ultimateefficiency of the chromatographic system. For example, in achromatographic system using 4.7 mm column tubing and a mobile phaseflowing at 1-2 mL/min, tubing having an outer diameter of 1/16 inch andan inner diameter of about 0.010 inch is typically used to plumbconnections between the various HPLC components (e.g. pump, injector,column, and detector). For these flow rates and tubing dimensions, it isrelatively easy to machine port details to tolerances that will ensureminimal band broadening at tubing interfaces.

A desire to reduce mobile-phase solvent consumption, in part, hasmotivated a trend towards reducing column inner diameter. Thus, severalscales of chromatography are now commonly practiced; these are typicallydefined as shown in Table 1 (where ID is inner diameter.)

TABLE 1 HPLC Scale Column ID Typical Flow range Analytical 4.7 mm 1smL/min Microbore 1-2 mm 100s μL/min Capillary 300-500 μm 10s μL/min Nano50-150 μm 100s nL/min

Microbore HPLC has often been practiced with equipment similar to thatused for analytical scale HPLC, with minor modifications. Aside fromrequiring the exercise of a small degree of additional care in makingfittings, microbore HPLC typically requires an operating skill levelsimilar to that of analytical scale HPLC.

In contrast, capillary and nano-scale HPLC require relativelysignificant changes in HPLC components relative to analytical-scaleHPLC. Generation of stable mobile-phase flows of less than about 50μL/min is relatively difficult using standard open-loop reciprocatingHPLC pumps, such as those commonly found in analytical and microboreHPLC systems.

For capillary-scale chromatography, stainless-steel tubing is usable forcomponent interconnections; however, the inner diameter must typicallybe less than 0.005 inch (less than about 125 μm). Care is generallyrequired in the manufacture of fitting terminations to avoid creation ofeven minute amounts of dead volume.

For nano-scale chromatography, tubing having inner diameters of about25-50 μm is typically required to interconnect components of aninstrument (e.g., to connect a pump to a separation column). Becausestainless-steel tubing is typically unavailable in these dimensions,polyimide-coated fused-silica tubing is typically used. Althoughfused-silica tubing has excellent dimensional tolerances and very clean,non-reactive interior walls, it is fragile and can be difficult to workwith. In addition, interconnection ports should be machined to exactingtolerances to prevent even nanoliters of unswept dead volume.

While the primary motivation to replace analytical-scale HPLC withmicrobore-scale HPLC may be the desire for reduced solvent consumption,moving to capillary-scale and nano-scale chromatography can supportimproved detection sensitivity for mass spectrometers, in addition tofurther reducing solvent consumption, when, for example, flows of lessthan about 10 μL/min are used. Moreover, capillary-scale or nano-scalesystems are often the only options for the sensitive detection typicallyrequired for applications involving small amounts of available sample(e.g., neonatal blood screening).

Despite the advantages of capillary-scale and nano-scale chromatography,HPLC users tend to employ microbore-scale and analytical-scalechromatography systems. As described above, these systems typicallyprovide good reliability and relative ease-of-use. In contrast,maintenance of good chromatographic efficiency while operating acapillary-scale or nano-scale chromatographic system requiressignificant care when plumbing the system (e.g., using tubing to connectpump, injector, column, and detector).

In practice, an operator switching from an analytical or microbore-scalesystem to a capillary or nano-scale system at times finds that betterseparation efficiency was achieved with the higher-flow rate (i.e., theanalytical or microbore-scale) system. This typically occurs due toinsufficiency in the operator's knowledge or experience required toachieve low band-spreading tubing interconnections. Moreover, use ofsmaller inner-diameter tubing at times can lead to frequent plugging oftubing.

Due the relative difficulty typically encountered with capillary-scaleHPLC systems and, even more so, with nano-scale HPLC systems, suchsystems have primarily been used only when necessary, such as for smallsample sizes, and when a relatively skilled operator is available. Thus,analytical laboratories tend to possess more analytical-scale andmicrobore-scale systems than capillary-scale and nano-scale systems, anddo not realize the full benefits available from capillary-scale andnano-scale HPLC.

Proteomic analyses often utilize a trap column for sample enrichment andcleaning prior to separation of the sample in an analytical column.Often, different packing material chemistries are used for the trap andseparation columns; sample components trapped on the trap column may beserially driven from the trap to the separation column during agradient-based mobile phase elution process. The components can beinitially focused at the head of the analytical column, due to thedifferent chemistry, until the gradient attains a level that drives thecomponent from the chemistry of the analytical column. It is also commonto place the analytical column in an oven to provide a stable, elevatedtemperature, which promotes elution of sample components from theanalytical column.

As noted, above, analyses involving small sample volumes, such as avolume of 50 μL, present challenges, even in an apparatus configured fornano-analysis. In particular, it remains difficult, if not impossible,to remove all significant sources of sample dispersion when handlingsuch small sample volumes.

SUMMARY

Some embodiments of the invention arise, in part, from the realizationthat a trap-column-to-analytical-column temperature differential—inparticular, where the trap is elevated relative to the analyticalcolumn—potentially provides improved sample separations, in particular,for complex protein-related samples. Some embodiments of the inventionarise, in part, from the realization that small volumes ofsmall-molecule-based samples can be focused at the head of a separationcolumn, via cooling, to mitigate dispersion effects associated withupstream plumbing. Potential improvements include, for example, improvedsensitivity and resolution. Moreover, temperature manipulation isoptionally coordinated with trap-versus separation-column differences inchemistry and/or solvent-composition profiles to provide desirably sharpcomponent peaks that elute from a separation column.

Moreover, some embodiments arise, in part, from a realization that anintegrated high-pressure chemical-separation device, such as an HPLCapparatus or an ultra-high-pressure LC (UHPLC) apparatus, isadvantageously fabricated, in part, from sintered inorganic particles.Some embodiments of the invention provide nano-scale microfluidic LCinstruments that offer integration of a trap/enrichment column(s) and aseparation column(s) on one (or more) ceramic-based substrates; some ofthese embodiments include features that support cooling of theenrichment column to enhance cycle time and/or improve enrichment-columnperformance.

Accordingly, one embodiment features a method of analyzingprotein-related samples. The method includes: providing a complexprotein-related sample; loading a sufficient quantity of the sample ontoa trap column to overload the trap column; heating an analytical columnand heating the trap column to a greater temperature than the analyticalcolumn; and pumping a solvent, to the trap column, having a solventcomposition profile that, in cooperation with a temperaturedifferential, causes at least some of the components to elutesequentially from the trap column to the analytical column and focus onthe analytical column prior to eluting from the analytical column.

An alternative embodiment features an apparatus for chromatographicseparation of a sample. The apparatus includes: a trap column; aseparation column in fluidic communication with the trap column; atrap-column heater; a separation-column heater; a solvent pump unit; anda control unit. The control unit includes instructions that, whenimplemented, causes the apparatus to: load a sufficient quantity of acomplex protein-related sample onto the trap column to overload the trapcolumn; heat the analytical column and the trap column so that the trapcolumn has a greater temperature than the heated analytical column; andpump a solvent, to the heated trap column, having a solvent compositionprofile that, in cooperation with the temperature differential, causesat least some of the components to elute sequentially from the heatedtrap column to the heated analytical column and focus on the heatedanalytical column prior to eluting from the heated analytical column.

Another embodiment features a method of chemical analysis, whichincludes: (a) providing a ceramic-particle-based and/or metal-basedmicrofluidic substrate defining a trap column and an analytical columnin fluidic communication with the trap column; (b) loading a sample onthe trap column while the trap column is at a temperature in a loadrange; (c) heating at least a portion of the substrate containing theanalytical column to provide a temperature above ambient during elutionof the sample through the analytical column, such that the trap columnis incidentally heated; (d) pumping a solvent to the trap column toelute the sample components from the trap column to the analyticalcolumn at the temperature above ambient, causing the components to elutefrom the analytical column; (e) cooling at least a portion of thesubstrate containing the trap column, after elution of the componentsfrom the analytical column, to return the trap column to a temperaturein the load range; and (f) repeating (b) through (e) for each of one ormore subsequent samples.

An alternative embodiment features an apparatus for chromatographicseparation of a sample. The apparatus includes: a ceramic-particle-basedand/or metal-based microfluidic substrate defining a trap column and ananalytical column in fluidic communication with the trap column; afluidic conduit having an outlet disposed to direct a fluid towards alocation of the trap-column to cool at least a portion of themicrofluidic substrate; a separation-column heating unit disposed toheat at least a separation column portion of the microfluidic substrateduring separation of a sample; and a solvent pump unit for pumping asolvent composition to an inlet of the trap column.

Still another embodiment features a method of analyzing small molecules,The method includes: cooling at least a portion of an analytical columnproximate to an inlet of the analytical column; loading, onto the cooledportion of the analytical column, a nano-scale sample comprising aplurality of different small-molecule components; heating the analyticalcolumn to promote elution of the loaded components; and pumping asolvent, to the heated analytical column, to elute the components fromthe analytical column.

Some preferred embodiments entail mass analysis.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further advantages of this invention may be betterunderstood by referring to the following description in conjunction withthe accompanying drawings, in which like numerals indicate likestructural elements and features in various figures. The drawings arenot necessarily to scale, emphasis instead being placed uponillustrating the principles of the invention.

FIG. 1A is a block diagram of a portion of a chemical analysis device,in accordance with one embodiment.

FIG. 1B is a flowchart of a method of analysis of complex proteinsamples, in accordance with one embodiment.

FIG. 1C is a flowchart of a method of analyzing samples of smallmolecules, in accordance with one embodiment.

FIG. 1D is a flow chart of a method of chemical analysis using one ormore microfluidic substrates.

FIG. 2 is a front view of an embodiment of the liquid chromatographymodule.

FIG. 3 is a view of the liquid chromatography module with an open coverto show a clamping assembly housed within.

FIG. 4 is a side view of the clamping assembly.

FIG. 5 is a view of an embodiment of an end housing of the clampingassembly.

FIG. 6 is a view of the left side of the microfluidic cartridge.

FIG. 7 is a side view of the microfluidic cartridge with the right sideremoved, showing a push block superimposed upon the microfluidicsubstrate in the microfluidic cartridge.

FIG. 8 is a side view of the microfluidic cartridge with the left sideremoved.

FIG. 9 is a side view of one embodiment of a microfluidic substratewithin the microfluidic cartridge.

FIG. 10A is a view of an embodiment of a separation device having morethan one microfluidic substrate.

FIG. 10B is a cross-sectional view of a portion of the device of FIG.10A, illustrating, in more detail, fluidic-connection and alignmentfeatures.

DETAILED DESCRIPTION

As used herein, the term “complex protein sample” means a sample thatincludes at least hundreds of different proteins and/or peptides. Atypical complex sample, for example, includes approximately 10,000proteins, which may be digested, prior to chromatographic separation,yielding a complex protein sample that includes approximately 5 to 15peptides per protein in the complex sample; such a complex proteinsample will then, upon separation, yield thousands of chromatographicpeaks. Some embodiments of the invention provide sharp chromatographicpeaks for a complex protein sample, by stepping peptides, in sequence,off of a trap column, at least in part through manipulation of trapcolumn and separation column temperatures. Various methods, according tosome embodiments of the invention, are well suited to complex proteinsamples, as encountered, for example, in proteomics work.

As used herein, the term “small-molecule sample” means a sample thatcontains one or more different “small molecules”, which are organicnon-polymeric molecules. The term “small molecule” is used herein in aconventional sense; a common definition of a “small molecule”,especially as found in the field of pharmacology, is generallyrestricted to a molecule that binds with high affinity to a biopolymer,such as a protein, nucleic acid, or polysaccharide, and, in addition,alters the activity or function of the biopolymer. The upper molecularweight limit for a small molecule is taken to be approximately 800Daltons. The molecular-weight limit generally accommodates thepossibility of rapid diffusion of small molecules across cell membranes,permitting intracellular access and activity. This molecular-weightcutoff is generally accepted as a necessary but insufficient conditionfor oral bioavailability.

Small molecules can have a variety of biological functions, serving ascell signaling molecules, as tools in molecular biology, as drugs inmedicine, as pesticides in farming, and in many other roles. Thesecompounds can be natural (such as secondary metabolites) or artificial(such as antiviral drugs); they may have a beneficial effect against adisease (such as drugs) or may be detrimental (such as teratogens andcarcinogens).

Biopolymers such as nucleic acids, proteins, and polysaccharides (suchas starch or cellulose) are generally consider as not falling within thedefinition of small molecules, although their constituent monomers—ribo-or deoxyribonucleotides, amino acids, and monosaccharides,respectively—are often considered to be small molecules. Very smalloligomers are also usually considered small molecules, such asdinucleotides, peptides such as the antioxidant glutathione, anddisaccharides such as sucrose.

The term “temperature differential” is used herein to mean that two, ormore, entities, or portions of the same or different entities, havedifferent temperatures. A “temperature differential” refers to thetemperature difference and/or the particular absolute temperatures ofthe objects having different temperatures.

Various non-limiting embodiments of the invention relate, preferably, tochromatographic separations of complex protein samples or small volumesof small-molecule samples. These embodiments provide sharperchromatographic peaks through manipulation of column(s) temperature.Some embodiments include enrichment and separation columns, or onlyutilize one or more separation columns. Some embodiments, which in somecases are configured for separations of small amounts of sample, utilizeceramic-based or other suitable substrates, such as a bonded-titaniumsubstrate. In view of this description, other embodiments and componentswill be apparent to one having ordinary skill in the compound-analysisarts.

Some embodiments include features that provide cooling and/or heating ofone or more enrichment columns and/or cooling and/or heating of one ormore separation (e.g., analytical) columns. In some cases, temperaturecontrol supports improved enrichment-column performance and shortercycle time of sample analyses. In some preferred embodiments, atemperature differential, between a trap column and an analyticalcolumn, is provided during elution of a sample. In particular, a trapcolumn, holding an enriched sample, is preferably held at a higherelevated temperature than the elevated temperature of an analyticalcolumn.

Some embodiments of the invention are based on microfluidic substrates;in the following, more general embodiments are described first, followedby examples that are specific to microfluidic substrates. In thefollowing, the term “analytical column” is used interchangeably with“separation column”, for convenience, and is not intended to limit allembodiments of the invention to columns of any particular dimensions,width or diameter, to any particular flow rates, or to any particularsample-volume sizes.

In some embodiments, a trap column is overloaded; during overloading,the band of enriched components grows broad as active sites are used up.Preferably, the broad bands of components are caused to focus or narrowas they move onto an analytical column. Various embodiments of theinvention utilize temperature and/or chemistry variations to obtaindesired focusing, sensitivity and/or resolution. Some preferredembodiments relate to protein or small-molecule analyses.

Regarding proteomics, some embodiments entail gradient elution, such asthrough the use of water and acetonitrile; as known in the art,different classes of peptides are generally released from a packingmedium at different concentrations. After enrichment on a trap column,the release of a big band of a component from the trap can cause the bigband to gradually enter the analytical column. Various embodimentsprovide focusing of such broad component bands into narrow bands in thevicinity of the head of the analytical column.

Some embodiments, in combination with temperature manipulation, utilizedifferent chemistries in different columns. Some of these embodimentsprovide capillary- or nano-scale chromatography. For example, ananalytical column optionally includes 1.7 μm diameter ethylene bridgedhybrid particles (such as an ACQUITY UPLC® BEH TECHNOLOGY™ C18 column of75 μm inner diameter and 100 mm length, and 1.7 μm particle size,available from Waters Corporation, Milford, Mass.) while a trap columnoptionally includes 5 μm diameter silica particles (such as ananoACQUITY UPLC® 10K SYMMETRY® C18 column of 180 μm inner diameter and20 mm length, available from Waters Corporation.) Using such columns, asample component can be caused to come off the trap column at, forexample, a lower acetonitrile strength than required for elution fromthe analytical column; the component will thus tend to form a narrowband on the analytical column prior to elution at a time when thesolvent strength has further increased.

Some preferred embodiments of the invention thus use a chemistrymismatch with temperature manipulation and mismatching to obtain sharpcomponent peaks, good sensitivity for substantially all components in asample, and/or efficient analyses. As will be apparent to one of skill,in view of the description provided herein, solvent type and temporalcomposition profile are suitably chosen in cooperation withcolumn-temperature manipulation and, optionally, chemistry manipulation,to support analysis of a particular type of sample. One of skill willalso understand that the following examples merely illustrate variousaspects of the invention, and are not intended to limit all embodimentsto a specific collection of features.

FIG. 1A is a block diagram of a portion of a chemical-analysis device500, which is used to illustrate some broad principles of some methodsand apparatus, according to some embodiments of the invention. Thedevice 500 includes a separation column 510, a thermal unit 511 inthermal communication with the column 510 for heating and/or cooling ofall or portion(s) of the column 510, an optional cooler 512 in thermalcommunication with at least a portion of the separation column 510proximate to an inlet of the column 510, an optional trap column 520,having an outlet in fluidic communication with an inlet of theseparation column 512, an optional trap thermal unit 521 in thermalcommunication with the trap column 521, for heating and/or cooling ofall or portion(s) of the trap column 520, and an optional control module530 to control various operations of the device 500 such as heatingand/or cooling by the thermal units 511, 521 and the cooler 512.

The device 500 is optionally used to implement various embodiments ofmethods of sample separation. For example, FIG. 1B is a flowchart of amethod of analysis 600 of complex protein samples, in accordance withone embodiment of the invention. The method 600 includes loading 610 asufficient quantity of a complex-protein sample onto a trap column tooverload the trap column, heating 620 an analytical column, heating 630the trap column to a greater temperature than the analytical column,thus providing a temperature differential between the trap andanalytical columns; and delivering 640 a mobile phase having a solventcomposition profile that, in cooperation with the temperaturedifferential, causes at least some of the overloaded components to elutesequentially from the trap column to the analytical column and focus onthe analytical column prior to eluting from the analytical column.

As one example, the delivered 640 mobile phase is based on a temporalgradient of acetonitrile/water, which serves to release differentpeptide components in sequence from the trap column and later, insequence, from the analytical column. Preferably, the components arereleased from the trap column at a lower acetonitrile strength than theacetonitrile strength required to release the same components from theanalytical column. Thus, a physically broad band of a component can bereleased from the trap column and become a physically narrow band on theanalytical column, prior to release from the analytical column when asuitably high concentration of acetonitrile arrives at the analyticalcolumn.

As illustrated by the method 600, some preferred embodiments supportanalyses of complex protein samples through thermal manipulation oftrap/enrichment columns and separation/analytical columns. The methodsare particularly well suited to chromatographic analyses samplequantities that will tend to overload an enrichment column.

As understood by one of skill in the protein liquid chromatograph arts,such a sample will tend to load a broad band of sites of an enrichmentcolumn. Through some prior approaches of then passing the overloadedsample to a separation sample, broad bands of components eluting fromthe enrichment column tend to produce broad peaks eluting from theseparation column. Thermal manipulation, however, according to someembodiments of the invention, promotes sequential elution from theenrichment column at solvent compositions that permit temporary bindingat sites on the analytical column prior to subsequent elution from theanalytical column as the solvent composition profile progresses.

Since sample components arrive at the analytical column in sequence, thecomponents have a greater portion of binding sites available to themthan in the case of the overloaded enrichment column. Thus, a broadcomponent band, arriving at the analytical column can bind in a narrowband near the head of the analytical column, and elute as a relativelysharp band/peak from the analytical column. In this manner, someembodiments provide relatively good chromatographic resolution for largecomplex protein samples.

Moreover, as indicated above, different stationary-media chemistries areoptionally selected to promote sequential elution of sample componentsand focusing on an analytical column of broad component bands elutingfrom an enrichment column. Thus, thermal manipulation and chemistrymanipulation can cooperate with solvent profile selection to providegood chromatographic resolution.

Loading 610 of an enrichment column is optionally assisted by providingthe column with an ambient or sub-ambient temperature. Thus, optionally,the trap column is allowed to equilibrate with a room temperature of,for example, 25° C., or is cooled to a sub-ambient temperature, as lowas, for example, −5° C. Cooling is optionally used to increase thehydrophobicity of the trapping medium, to promote capture of the sampleon the trap column. After loading 610, the trap column is heated 630 toa greater temperature than the analytical column. Preferably, duringelution from the trap, the analytical column has greater hydrophobicitythan the trap column, as provided, for example by temperature andchemistry differentials.

Preferably, as discussed, during elution, the trap is set at a highertemperature than the analytical column. For example, the trap is heated630 to a temperature in a range of approximately 45° C. to 65° C. orgreater, while the analytical column is heated 630 to approximately 35°C., during an elution phase.

For heating 630 of the analytical column, the analytical column isoptionally maintained at a substantially constant temperature while atrap column alternates between relatively low and high temperatures,respectively, for alternating enrichment 610 and elution 640 steps of ananalysis process. Thus, for example, during loading 610, the columnshave temperatures of 20° C./35° C., and during elution 640, the columnshave temperatures of 45° C./35° C.

A temperature differential is chosen, for example, to provide optimumnarrowing of component bands, as relatively broad bands of componentsarrive at, and are temporarily retained on, the analytical column. Moregenerally, enrichment trap chemistry and/or temperature are selected toprovide trapping of substantially all components, or all desiredcomponents, of a protein-related sample. For efficient elution with goodpeak resolution, the chemistries and/or temperature and/or temperaturedifferential are selected for the protein-related sample of interest.

During trapping 610, too high a temperature of the trap column can causeloss of compounds that exhibit little or no retention at the too hightemperature; as one alternative, an enrichment temperature is chosensuch that it is cool enough so that substantially all compounds, or allcompounds of interest, are trapped. Preferably, the trap temperature isthan increased for the elution 640 phase. Use of a same trap temperatureduring elution 640 potentially inhibits refocusing on the analyticalcolumn.

For example, a cool trap can require a high acetonitrile concentrationto remove compounds from the trap. Such a concentration can be too greatto provide desired refocusing on the analytical column; in particular,if the analytical column is at a higher temperature than a cool trap,compounds may run straight through the analytical column, retainingbroad peak widths as developed on the trap column.

As mentioned, some embodiments employ both selection of differentchemistries and different temperatures to improve focusing and othercharacteristics; alternative embodiments use the same materials anddifferential heating only. Generally, a certain elevation of temperaturelowers the percentage of acetonitrile at which a peptide will elute.

As one particular example, illustrative of some proteomics work, ananalytical column having a relatively small inner diameter of 75 μm isused for analysis of a large sample volume. A trap column having aninner diameter of 180 μm is used, for example, to concentrate and purifythe sample by, in part, washing salts from the loaded sample, and toprovide quicker loading by use of the relatively wide diameter. Areference-protein sample, suitable for testing performance, is analyzedwith a mobile phase composed of a varying mixture of mobile phase A(0.1% formic acid in water) and mobile phase B (0.1% formic acid inacetonitrile.) The trap column is a nanoACQUITY UPLC® 10K Symmetry® C18180 μm×20 mm, 5 μm particles (available from Waters Corporation,Milford, Mass.)

The trapping flow rate is 10 μL/min, the trapping composition is 99.5%mobile phase A/0.5% mobile phase B, the load time is 1.5 min, the linearvelocity is 14.89 mm/sec, the analytical column is a nanoACQUITY UPLC™®BEH TECHNOLOGY™ C18 column of 75 μm diameter×100 mm length and packedwith 1.7 μm particles (available from Waters Corporation, Milford,Mass.), the flow rate is 0.300 μL/min, the gradient is 5% mobile phase Bto 40% mobile phase B in 30 minutes, the sample is 100 fmol/μL ofenolase in 0.1% formic acid in 97/3 water/acetonitrile, the injectionvolume is 2 μL in a “partial loop mode” for a 200 fmol load of enolase,a lock mass solution is 500 fmol GFP in 0.1% formic acid in 97/3water/acetonitrile, and a lock mass flow rate is 0.5 μL/min.

The eluent, in this example, is delivered to a mass-spectrometryinstrument portion of the apparatus. The example instrument is a Q-T ofMicro™ (available from Waters Corporation, Milford, Mass.), the scanrange is 400-1700 m/z, the scan time range is 0-40 minutes, the scantime is 0.88 sec, and the inter-scan delay is 0.1 sec.

A sample of 100 fmol/μL enolase in 0.1% formic acid in 97/3water/acetonitrile may be prepared by diluting a stock of 1 pmol/μLenolase in 0.1% formic acid in 70/30 water/acetonitrile ten times. Inone example, a 2 μL injection of the sample is made for enrichment, withthe trap column approximately at or below ambient temperature. The trapand analytical columns are then both heated, the trap to a highertemperature than the analytical column, and the enriched sample iseluted through the trap and analytical columns. The trap is then cooledto prepare for enrichment of the next sample.

Some embodiments exploit temperature control features with otherfeatures that serve to reduce plumbing-related dispersion. For example asubstrate having adjacent trap and separation columns (see FIG. 9) isoptionally packed with trap and separation materials that are contiguouswith one another.

As mentioned above, sample enrichment is utilized for any one or more ofseveral reasons, for example: to limit injection volume, which can helpto present a narrow band to the analytical column; to remove salts orother undesired components before passing a sample to an analyticalcolumn and then, potentially, to a mass spectrometer; and to morerapidly analyze large sample volumes (the trap column optionally has alarger inner diameter and lower back pressure than the analyticalcolumn.)

Generally, though a sample may not overload the analytical column, itmay still overload a trap column, where, for example, the sample is acomplex mixture, all components of which are simultaneously placed onthe trap. Since components of the sample elute serially from the trap,the analytical column is potentially not overloaded. Thus, for example,the mass load of a fraction entering the analytical column does notoverload the analytical column because the fraction is insufficient tosaturate all of the active sites of the analytical column.

Some embodiments of the invention relate to small-molecule analyses,and, preferably, small samples sizes of such samples. Such samplesgenerally do not require enrichment or cleaning, prior to separation,but can exhibit physical broadening of component bands due to dispersionas the sample travels, for example, from an injector to the head of ananalytical column.

FIG. 1C is a flowchart of a method of analyzing samples of smallmolecules, in accordance with one embodiment of the invention. Themethod includes cooling 710 at least a portion of an analytical columnproximate to an inlet of the analytical column, loading 720, onto thecooled portion of the analytical column, a nano-scale sample ofdifferent small-molecule components, heating 730 the analytical columnto promote elution of the loaded components, and pumping 740 a solvent,to the heated analytical column, to elute the components from theanalytical column. Preferably the analytical column has an innercross-sectional dimension in a range of approximately 150 μm toapproximately 500 μm. The cooled portion of the analytical column can beimplemented as a distinct column connected to a primary analyticalcolumn, but suitably disposed relative to the primary analytical columnto provide little or no dispersion of a sample as it travels from thedistinct column to the primary column.

Some small-molecule samples, as will be understood by one of ordinaryskill, will be relatively clean, and can be directly separated withoutfirst cleaning. The method 700 is optionally implemented with a singlecolumn, for both loading 720 and separation 740. Some embodiments entailcapillary-scale chromatography; preferably, some embodiments entailcapillary-scale chromatography, using, for example a 300 μm innerdiameter analytical column. The associated small sample volumes (forexample, sample injections of 100's of nL or less) can lead tosignificant dispersion in the plumbing that is common in typicalliquid-chromatography apparatus. The method 700 provides a solution todifficulty in transferring a well controlled injection plug from aninjector to the head of an analytical column.

Even a significantly dispersed injection plug can be sharpened on thehead of an analytical column, through application of some embodiments ofthe invention. After focusing of a sample plug, the temperature is thenraised to a level desired for separation and elution from an analyticalcolumn. The head only, or larger portions of a column, or the entirecolumn is optionally cooled during loading 720.

Alternatively, the small-molecule sample is focused on a cooled column,which then releases the focused sample to an analytical column that isconnected to the cooled column in a manner to preserve the focused plug.

Some embodiments of the invention entail microfluidic components, suchas substrate(s) that contain column(s) and/or conduits and/or othermicrofluidic features. For example, FIG. 1D is a flow chart of a method800 of chemical analysis using a ceramic-particle-based and/ormetal-based microfluidic substrate(s) defining a trap column and ananalytical column in fluidic communication with the trap column. Themethod includes: loading 810 a sample on the trap column while the trapcolumn is at a temperature in a load range; heating 820 at least aportion of the substrate containing the analytical column to provide atemperature above ambient during elution of the sample through theanalytical column, wherein the trap column is incidentally heated;pumping 830 a solvent to the trap column to elute the sample componentsfrom the trap column to the analytical column at the temperature aboveambient, causing the components to subsequently elute from theanalytical column; cooling 840 at least a portion of the substratecontaining the trap column, after elution of the components from theanalytical column, to return the trap column to a temperature in theload range; and repeating 850 steps 810, 820, 830, 840 for each of oneor more subsequent samples.

The analytical column preferably has particles having a greaterhydrophobicity than particles of the trap column. The temperature aboveambient of the heated analytical column is preferably approximately 35°C. or greater. The analytical column optionally has an innercross-sectional dimension in a range of about 50 μm to about 150 μm, forexample, for protein analysis.

A microfluidic device, according to various embodiments of theinvention, can take a variety of forms. The device can include more thanone substrate; columns can be disposed on different substrates tofacilitate control of temperature of the columns, for example, torestrict flow of heat from one column to the other, and/or, similarly,to provide different columns with different temperatures. A singlesubstrate optionally includes features, such as thermal breaks, torestrict heat flow. For example, a substrate optionally includes acut-out portion and/or portions of a relatively low thermal-conductivitymaterial.

As noted above, some preferred embodiments of the invention entailHigh-Performance Liquid Chromatography (HPLC) instruments. As describednext, some of these embodiments utilize microfluidic component(s). Theherein described examples of HPLC embodiments having microfluidicfeatures have an installation chamber for receiving a microfluidiccartridge having an electrospray emitter, and for bringing the tip ofthe emitter into operable communication with mass spectroscopycomponents of the HPLC instrument. The microfluidic cartridge houses oneor more microfluidic substrates; a substrate is preferably asubstantially rigid, ceramic-based, multilayer microfluidic substrate(also referred to herein as a ceramic tile), for example, as describedin US Patent Publication No. 2009/032135, Gerhardt et al., which isincorporated herein by reference. For protein samples, the ceramic ispreferably a High-Temperature Co-fired Ceramic (HTCC), which providessuitably low levels of loss of sample due to attachment of sample towalls of conduits in the substrate. Some embodiments dispose separationcolumns(s) and trap column(s) in different substrates.

A channel, formed in the layers of the substrate, operates, for example,as a separation column. Apertures in the side of the substrate—formed,for example, via laser etching—provide openings into the channel throughwhich fluid may be introduced into the column. Fluid passes through theapertures under high pressure and flows toward the electrospray emittercoupled at the egress end of the channel. Holes in the side of themicrofluidic cartridge provide fluidic inlet ports for delivering fluidto the substrate. Each of one or more fluidic inlet ports align with andencircle one of the fluidic apertures.

A clamping mechanism applies a mechanical force to one side of themicrofluidic cartridge, urging the substrate against fluidic nozzlescoupled to the installation chamber. The nozzles deliver fluid to thesubstrate through the fluidic inlet ports of the cartridge.

In addition to the substrate, embodiments of microfluidic cartridgespreferably house internal circuitry and one or more temperature controlunits for heating and/or cooling the substrate(s), such as one or moreportions of the substrate(s). In some embodiments, an aperture in themicrofluidic cartridge housing provides a window through which pogo pinssupply low voltage and other electrical signals to internal circuitry.Another aperture in the microfluidic cartridge housing optionallyprovides access for a cooling gas, directed at one or more substrates.

Other embodiments of the invention entail apparatus and methods thatmanipulate the temperature of tube-based columns, for exampleconventional separation columns and/or trap columns for improvedseparations. Preferred embodiments are applied to complex proteinsamples or small-molecule samples, in particular, small volume samples.The following described examples will be understood by one of skill tomerely illustrate some optional implementations of the invention, whilenot limiting all implementations to any particular collection offeatures.

FIG. 2 shows a front view of an embodiment of a liquid chromatographymodule 12 having a housing 20. The housing 20 has an upper section 22with an on-off switch 23 and with status and pressure indicators 24, amiddle section 26 having a slot 14 for receiving the microfluidiccartridge and an arm portion 28, and a lower section 30 having anadjustment knob 32 extending from an opening in the housing. Theadjustment knob 32 is coupled to an interior x-translation stage (partlyvisible) for moving a microfluidic cartridge along the x-axis.Adjustment knobs for y-translation and z-translation stages (not shown)also enable y-axis and z-axis adjustments of the position of themicrofluidic cartridge relative to an mass spectroscopy (MS) (notshown.) Such adjustments provide, for example, positioning of an emittertip outlet in relation to an MS inlet orifice.

Coupled to the arm portion 28 is a lever 34 that is rotatable about apivot point 36 between a clamped position and an unclamped position. InFIG. 2, the lever is in the unclamped position. Counterclockwiserotation of the lever about the pivot point, approximately 180 degrees,moves the lever into the clamped position. At one end of the housing, anelectrical cable 38 and an electrical signal conductor 40 enter thehousing through an opening 42 in the front of the housing. Theelectrical cable 38 supplies a high voltage, and the electrical signalconductor 40 supplies a low voltage, to the microfluidic cartridge, asdescribed herein. Not shown are the microfluidic tubing and one or moregas lines, which also enter the housing through the opening 42, forbringing fluid and gas, respectively, to the microfluidic cartridge.

FIG. 3 shows the housing 20 with its front cover 50 opened to expose aclamping assembly 60 within. A hinge along a base edge of the housingattaches the front cover 50 to a translation stage support 52. Thetranslation stages and adjustment knobs are absent from the drawing tosimplify the illustration. Other components residing within the housinginclude a circuit board 62. The translation stage support 52, clampingassembly 60, and circuit board 62 are coupled to a rear panel 64 of thehousing.

The electrical cable 38 and an electrical conduit 66 couple to one sideof the clamping assembly 60. The electrical cable carries a high voltage(e.g., 3000 volts), and the electrical conduit 66 bundles a plurality oflow-voltage electrical conductors. Not shown are the microfluidic tubingand gas line that are also coupled to the same side of the clampingassembly 60 as the electrical cable 38 and electrical conduit 66.

The clamping assembly 60 has a slot 68 for receiving a microfluidiccartridge and a post 70 to which the lever 34 (FIG. 2) is attached. Whenthe front cover 50 is closed, the slot 14 in the front cover 50 alignswith the slot 68 of the clamping assembly 60, the adjustment knob 32 ofFIG. 2 (not shown in FIG. 3) projects through the opening 72 in thefront cover 50, and the post 70 projects through another opening, whichis obscured by the sidewall of the arm portion 28.

FIG. 4 shows a side view of the clamping assembly 60, with amicrofluidic cartridge 16 passing through both slots in sidewalls 84 ofthe clamping assembly 60. The clamping assembly 60 has a body 80 coupledto an end housing 82. The arm 110 catches a nook in an upper edge of themicrofluidic cartridge 16, preventing the microfluidic cartridge fromsliding further through the slots 68. A high-voltage electrical cable 38couples to the opening 106 and an electrical conduit 66 couples to atwo-piece bracket 92 of a pogo-pin block. In addition, a coupler 112connects to the opening 104 in the L-shaped retainer 100.

FIG. 5 shows an interior view of one embodiment of the end housing 82,including opposing sidewalls 84-1, 84-2, separated by back wall 86. Thesidewall 84-1 has the slot 68-1, and the sidewall 84-2 has the slot68-2. Installed in the back wall 86 are pogo pin block 88 and a fluidicblock 90.

The interior side of the pogo pin block 88 has a recessed region 140with a pogo pin electrical connector 142 projecting inwardly from asurface thereof. In this example, the electrical connector 142 has tenelectrically conductive pogo pins 144 for conducting electrical signals.Each pogo pin 144 is an individual cylindrical, spring-loaded electricalconductor for transmitting electrical signals.

The interior side of the fluidic block 90 has a plurality ofmicrofluidic nozzles 130-1, 130-2, 130-3 (generally, 130) projectingtherefrom. In this embodiment, the nozzles 130 are three in number andarranged in a triangular pattern. The locations of these nozzles 130 arefixed with respect to each other. Fluid delivered by microfluidic tubesattached to the exterior side of the fluidic block 90 exits through oneor more of these nozzles 130. Situated below the triangular pattern ofnozzles 130, aligned with the nozzle at the apex of the triangle, is aguide pin 128 for guiding a cartridge.

The fluidic block 90 also has a coolant nozzle 131, for delivery of acoolant. For example, a N₂ delivery line is connected to the nozzle 131,or integral to the nozzle 131. To implement the above-described methods,a flow of N₂ is optionally directed at a substrate, via the nozzle 131,to cool the substrate, as desired. A temperature of the coolant isoptionally controlled, as is, optionally, the flow rate and/or velocity,to achieve a desired cooling effect of the substrate, as determined, forexample, theoretically and/or empirically.

Referring next to FIGS. 6-8, the microfluidic cartridge 16 works inconjunction with the fluid- and electrical-related components describedabove with regard to FIG. 4 and FIG. 5. As described below, in moredetail, the microfluidic cartridge 16 houses an emitter, a microfluidicsubstrate, a heater, and circuitry, and operates as an electromechanicalinterface for the delivery of voltages, electrical signals, and fluids(gas and liquid) to the various components housed within themicrofluidic cartridge 16.

This non-limiting example implementation of a microfluidic cartridge 16includes a housing made by joining two casing sections 200-1, 200-2, forexample, by snapping the halves together, or using glue or mechanicalfasteners, or any combination thereof. The two casing sections are alsoreferred to herein as the left and right sides of the microfluidiccartridge 16, with the terms left and right being determined by theorientation of the microfluidic cartridge 16 when it is inserted intothe clamping assembly 60. It is to be understood that such terms asleft, right, top, bottom, front, and rear are for purposes ofsimplifying the description of the microfluidic cartridge 16, and not toimpose any limitation on the structure of the microfluidic cartridge 16itself.

The right casing section 200-1 has a grip end 202 and an emitter end204. A curved region 206 within the grip end 202 provides a finger holdby which a user can grasp the microfluidic cartridge 16 when insertingand removing it from the liquid chromatography module 12.

The right casing section 200-1 has a rectangular-shaped window 208,within which resides a push block 210. The surface of the push block 210lies flush with the surface of the right casing section 200-1. Asdescribed further below, the push block 210 is not rigidly affixed tothe right casing section 200-1, and can move slightly in, out, up, down,left, or right; that is, the push block 210 floats within the window208. In one embodiment, the push block 210 is made of metal.

Disposed below the push block 210 is an opening 212, which extendscompletely through both casing sections 200-1, 200-2. Hereafter, theopening 212 is referred to as a through-hole 212. At the emitter end 204is a nook 214 in the top edge of the microfluidic cartridge 16. Withinthe nook 214, a movable fin 216 projects through the top edge betweenthe casing sections 200-1, 200-2.

FIG. 6 shows the left casing section 200-2 of the microfluidic cartridge16. Like the right casing section 200-1, the left casing section 200-2has a grip end 202 with a curved region 206 and an emitter end 204.Approximately central in the length of the left casing section 200-2 arethree nozzle openings 220 in a triangular pattern that matches thetriangular arrangement of the nozzles 130 of the fluidic block 90 (FIG.5.) The triangular pattern is desirable, to define a circular loadpattern. The center of the circle is preferably aligned to the axis ofmotion of a plunger (not shown) of the clamping assembly 60 (FIG. 6) toprovide a load balance on the metal-plate bosses 260 of the push block210; when the clamping assembly 60 is closed, the plunger pressesagainst the push block 210, which, in turn, presses against thesubstrate, which, in turn, presses against the nozzles 130 of thefluidic block 90. A third fluidic port is used, for example, for acalibration port; an operator can run a calibration fluid. The other twoports provide access to, for example, the analytical column and the trapcolumn.

Concentrically located behind each nozzle opening 220 is a microscopicfluidic aperture in the side of a microfluidic substrate housed withinthe microfluidic cartridge. The fluidic conduits of the microfluidicnozzles 130 of the fluidic block 90 have much larger inner diametersthan the size of the microscopic apertures in the substrate, whichfacilitates alignment therebetween. In one embodiment, each microscopicfluidic aperture has a 0.003″ square cross section, and eachmicrofluidic nozzle 130 has a 0.013″ orifice (lumen with a circularcross section) that aligns with and circumscribes the microscopicfluidic aperture on the substrate, such as a 0.003″ via (aperture with asquare cross section.)

The microfluidic nozzles 130 utilize a polymer-to-ceramic interface,relying only on the compressive stress provided by the clamping assembly60 (FIG. 6) to provide a fluidic seal; that is, the clamping assembly 60provides a greater pressure at the polymer-to-ceramic interface than theoperating fluidic pressure. For example, for an operating fluidicpressure of 5,000 psi—or alternative pressures, such as 15,000 psi—areimplemented with a clamping load of 130 pounds across the total surfacearea of the nozzle-to-substrate interface, producing an effectivefluidic seal for the selected operating pressure.

The left casing section 200-2 also has a coolant-nozzle opening 221,central to the three nozzle openings 220, and disposed to receive thecoolant nozzle 131. In this example, when the cartridge 16 is clamped inan operating position, the coolant nozzle 131 is disposed with itsoutlet proximate to the surface of the substrate(s) in the cartridge 16.

A coolant is optionally used, for example, to enhance sample enrichmentand/or sample focusing and/or decrease sample cycle time. For example, anano-flow apparatus is optionally utilized with a solvent gradient thatlasts for approximately 1.5 hours. The separation-column portion of asubstrate is optionally maintained at a temperature of 45° C. duringseparation. Once a sample run is completed, a coolant gas is optionallydirected at the substrate, as described above, to provider quickercooling to support quicker sample turnaround. For example, a gas flowoptionally sufficiently cools a substrate in approximately 2 minutes,which would often be satisfactory relative to a sample run time of 90minutes.

Directly above the apex of the triangularly arranged nozzle openings 220is a rectangular depression 222 within the left casing section 200-2.The depressed region 222 surrounds a rectangular-shaped window 224through which an array of electrical contacts 226 is accessed. Theelectrical contacts 226 are electrically conductive pads for makingelectrical contact with the pogo pins 144 of the pogo pin block 88 (FIG.5). The array of electrical contacts 226 is part of a flex circuitoverlaid upon the microfluidic substrate, as described further below inconnection with FIG. 13.

At the emitter end 204, the left casing section 200-2 has a gas inletport 225 for receiving a gas nozzle and a high-voltage input port 228for receiving the tip (pogo-pin) of the high-voltage electrical cable 38(FIG. 4). A plurality of holes 234 hold alignment pins 236 that are usedto align the casing sections 200-1, 200-2 when the halves are beingjoined.

The left casing section 200-2 further includes a rectangular-shapedgroove 230 along its bottom edge. The groove 230 has an open end 232 atthe emitter end 204, extends laterally therefrom, and terminates at thethrough-hole 212 situated below the nozzle openings 220. In addition,the groove 230 receives the guide pin 128 (FIG. 5) when the microfluidiccartridge 16 is inserted into the slot 68 (FIG. 4) of the clampingassembly 60. When the guide pin 128 reaches the through-hole 212, thenthe microfluidic cartridge 16 is fully installed in the chamber 120 andin position for clamping.

The substrate 250 is optionally formed in the following manner. Fivegreen-sheet layers, for multiple substrates 250, are pressed together,after desired patterning. Vias for fluidic apertures are laser etched inone or both sides of the pressed sandwich. Edge portions are defined bylaser etching. After firing, individual substrates 250 are snappedapart. Edges, or portions of edges, are optionally polished.

FIG. 7 shows a right side view of the microfluidic cartridge 16 with theright casing section 200-1 removed to reveal various components housedwithin and to show various features of the interior side of the leftcasing section 200-2. The various components include a microfluidicsubstrate 250 and a shutter 254, both of which are coupled to the leftcasing section 200-2 as shown.

A flex-circuit assembly 258 is folded over a top edge of themicrofluidic substrate 250, and includes the array of electricalcontacts 226. As described with respect to FIG. 6, these electricalcontacts 226 are accessible through the window 224 in the left casingsection 200-2.

The shutter 254 has a fin 216 (FIG. 8), and has an extension 217 thatprotects an emitter tube 264 by partially enveloping the tube 264, whenthe cartridge is not installed in the clamp assembly 60. The emittertube 264 is part of an emitter assembly that is described, in moredetail, below.

The flex-circuit assembly 258 includes a control circuitry portion 257and a heater portion (hereafter, heater 270). As noted, the flex-circuitassembly 258 folds over a top edge of the microfluidic substrate 250 andcovers a portion of the opposite side of the microfluidic substrate 250.An integrated circuit (IC) device 272 is mounted on the controlcircuitry portion of the flex-circuit assembly 258. In one embodiment,the IC device 272 is a memory device (e.g., EPROM) for storing programcode and data. The heater 270 covers a separation column within themicrofluidic substrate 250. Mounted to the heater 270 is a temperaturesensor 274.

The flex-circuit assembly 258 is constructed of multiple stacked layers(e.g., three, four, or five). The polymer substrate of each layer holdsdifferent interconnectivity or circuitry. One of the layers containsresistive traces of the heater 270. Electrical contacts at the two endsof the resistive traces connect to two pads 259 on the control circuitryportion 257. Another layer of the flex-circuit assembly 258 has viasthat electrically contact the ends of the resistive traces, anotherlayer has contacts to connect electrically to electrical components 272,274, and still another layer has pogo-pin contact pads 226 (FIG. 13).Through the flex-circuit assembly 258, each of the electrical components272, 274 and resistive traces are electrically coupled to the contactpads 226. The gas inlet port 225 opens into a well 276 that channelsinjected gas into a gas tube that delivers the gas to the emitter end204.

FIG. 8 shows a view of the microfluidic cartridge 16 from the left, withthe left casing section 200-2 removed to reveal various componentshoused within and to show features of the interior side of the rightcasing section 200-1. The flex-circuit assembly 258, is wrapped, asnoted, on this side of the microfluidic substrate 250, and includes thearray of electrical contacts 226 illustrated in FIG. 6. In addition, themicrofluidic substrate 250 has a high-voltage input port 290 forreceiving the electrically conductive terminal of the high-voltageelectrical cable 38 (FIG. 4). The high-voltage input port 290 isdisposed near an egress end of a separation column within themicrofluidic substrate 250, described below in connection with FIG. 9.

The microfluidic cartridge 16 includes a spray unit 340, which supportselectrospray output of sample that has passed through the substrate 250.The spray unit 340 includes a retainer 341 attached to the substrate250, the emitter tube 264, a spring (not shown) that urges the tube 264against the substrate 250, and a retainer cap 342. The cap 342 isfixedly or removably attached, for example, via a snap or threads, tothe retainer 341. The spring is disposed within the cap 342, when thespray unit 340 is assembled for operation.

The interior side of the right casing section 200-1 includes a ridge 292of casing material that runs from the emitter end 204 and terminates atthe through-hole 212. When the casing sections 200-1, 200-2 are joined,the ridge 292 runs directly behind the groove 230 (FIG. 12) on theexterior side of the left casing section 200-2. The ridge 292 providesstructural support to the microfluidic cartridge 16. In addition, bybeing directly opposite the groove 230, the ridge 292 resists bending ofthe cartridge 16 by the guide pin 128 (FIG. 5) should a user prematurelyattempt to clamp the microfluidic cartridge 16 before the microfluidiccartridge 16 has fully reached the proper position. In addition, noportion of the microfluidic substrate 250 lies directly behind thegroove 230, as a precautionary measure to avoid having the guide pin 128bend the microfluidic substrate 250 in the event of a premature clampingattempt.

The interior side of the right casing section 200-1 provides the otherhalf of the gas well 276, the walls of which align with and abut thosedefining the well 276 on the left casing section 200-2. To enhance atight seal that constrains gas to within the gas well 276, a fastener orpin 296 (FIG. 7) tightens the connection between the casing sections atthe opening 298 adjacent the well 276.

FIG. 9 shows a left-side view of an embodiment of the microfluidicsubstrate 250. In brief overview, the microfluidic substrate 250 isgenerally rectangular, flat, thin (approx. 0.050″), and of multilayerconstruction. Formed within the layers of the microfluidic substrate 250is a serpentine channel 300 for transporting liquid. The microfluidicsubstrate 250 includes a trap region 302 and a column region 304. In theembodiment shown, the microfluidic substrate 250 has three microscopicfluidic apertures 306-1, 306-2, 306-3 (generally 306). One of thefluidic apertures 306-1 intersects the channel 300 at one end of thetrap region 302; another of the fluidic apertures 306-2 intersects thechannel 300 at the other end of the trap region 302. In this embodiment,the third fluidic aperture 306-3 is unused. Alternatively, the thirdfluidic aperture 306-3 can be used, for example, as a calibration port;an operator can run a calibration fluid. The channel 300 terminates atan egress end of the microfluidic substrate 250. The fluidic aperture306-2 at the “downstream” end of the trap region 302 is optionally usedas a fluidic outlet aperture, for example, during loading of the trapregion 302, and is optionally closed to fluid flow, for example, duringinjection of a loaded sample from the trap region 302 into the channel300.

The microfluidic substrate 250 also has a high-voltage input port 290(FIG. 8) and a pair of alignment openings 310-1, 310-2 (generally, 310)that each receives a peg that projects from an interior side of the leftcasing section 200-2. The alignment openings 310 help position themicrofluidic substrate 250 within the microfluidic cartridge 16. Thesize of the alignment openings 310, with respect to the size of thepegs, allows the microfluidic substrate 250 some play within themicrofluidic cartridge 16.

As described above, some embodiments of the invention including coolingfeatures. In addition to, or alternative to, the above-describedcoolant-fluid-related features, other embodiments of the inventionutilize alternative mechanisms to cool substrate(s) and/or portions ofsubstrate(s). Any suitable cooling mechanisms, including knownmechanisms, are optionally included. Some suitable mechanisms includePeltier devices, use two or more substrates, as described next, use heatsinks, and/or use heat pipes, such as a closed system that volatilizes aliquid disposed over the heat source and condenses the volatilizedliquid in a cooled area, provide heat transport potentially at the speedof sound. Moreover, a substrate itself is optionally configured withchannels to carry a coolant. Related to the multiple-substrate option,some substrates include a thermal barrier or gap, disposed in, ordefined by, the substrate, to help thermally isolate trap and enrichmentcolumns from one another.

Some embodiments include features to permit cooling and/or heating of atrap column and/or a separation column, for example, to implement someof the specific temperature ranges and temperatures of above-describedexamples.

Some embodiments include multiple microfluidic substrates. For example,rather than a single microfluidic substrate 250, the microfluidiccartridge 16 can house a plurality of interconnected microfluidicsubstrates. Some example embodiments, having multiple substrates, aredescribed next, with reference to FIGS. 10A and 10B.

FIG. 10A shows a portion of a device, according to one embodiment of theinvention, which has two (or more) fluidically connected microfluidicsubstrates 250-1, 250-2. The substrates 250-1, 250-2 include aseparations substrate (also referred to herein as a column tile orsubstrate) 250-2 and a trap tile (or load substrate) 250-1. Themicrofluidic substrates 250-1, 250-2 are coupled to each other viacouplers 322-1, 322-2, which are optionally aligned to ports in thesubstrates 250-1, 250-2 via fittings 325-1, 325-2, 326-1, 326-2(generally, fittings 325, 326.) The trap tile 250-1 has afluid-conducting channel 300-1 and the column tile 250-2 has a fluidconducting channel 300-2, which are connected via a coupler 322-2. Thelocations of the channels 300-1, 300-2 are illustrated, although one ofskill will understand that the channels 300-1, 300-2 are internal to thesubstrates 250-1, 250-2.

This example of a trap tile 250-1 has two or three fluidic apertures.Coupled about each fluidic aperture is a fitting 320-1, 320-2, 320-3(generally 320). The fitting 320-3 is optionally located at a dummyaperture; the dummy location may merely be used, for example,application of a clamping site. The fittings 320 serve to self-align thetips of the nozzles (e.g., nozzles 130 of FIG. 5) on the fluidics block90 when the microfluidic cartridge 16 is installed in the chamber, asdescribed in more detail below. These fittings 310 can be made of metal,plastic, ceramic, or any combination of these materials or othersuitable materials. To couple the fittings 320 to the substrate 250-1,they can be glued, fastened, fused, brazed, or a combination thereof,for example.

The tile 250-1 has a notch, or open spot 318, which, for example,provides access for a nozzle to directly contact an aperture of theseparations substrate 250-2. The aperture can be a dummy aperture, forexample, for substrates that do not require use of a fourth nozzlecontacting the substrate at the site of the dummy aperture. The notch318 optionally assists alignment, orientation and/or substrateidentifications, for example.

FIG. 10B shows a cross-section of the device of FIG. 10A, sliced throughthe trap tile 250-1 and column tile 250-2, two of the fittings 320-1 and320-2 and two couplers 322-1, 322-2 respectively associated with thefittings 320-1, 320-2. The channel 300-1 of the trap tile 250-1 extendsfrom the fitting 320-1 to the fitting 320-2. The couplers 322-1, 322-2(generally, 322) connect the trap tile 250-1 to the column tile 250-2.Each coupler 322 has an associated a pair of alignment fittings 325,326: one fitting 325-1, 325-2 on the underside of the trap tile 250-1,and the other fitting 326-1, 326-2 on the top side of the column tile250-2. The coupler 322-2 (as noted above) provides a channel (or lumen)324 for fluid passing through the channel 300-1 of the trap tile 250-1to reach the channel 300-2 of the column tile 250-2. The couplers 322can be made of metal, plastic, ceramic, or any combination of thesematerials and can be glued, fastened, fused, and brazed, or anycombination thereof, to the tiles 250-1, 250-2.

Preferably, however, in some embodiments, the couplers 322 are formed ofa deformable matter, and need not be permanently attached to eithersubstrate 250-1, 250-2; in some embodiments that have a swappable traptile 250-1, the couplers 326-1, 326-2 are fixedly attached to the columntile 250-2, to facilitate swapping of the trap tile 250-1. For example,a device optionally includes a cartridge having a housing with a slot, adoor, or other means to permit access to the cartridge for removableand/or insertion of substrate(s).

A deformable material is optionally any suitable material or materials,for example, a material similar to or the same as the material of thenozzles 130. Mechanical pressure alone is optionally used to provide afluid-tight seal between the trap tile 250-1, the couplers 322 and thecolumn tile 250-2, using, for example, a clamping device, such as theclamping device described below. Alignment-assisting features, such asthe fittings 320-1 and 320-2, are optionally included in the embodimentillustrated in FIG. 9 and in other embodiments, to align nozzles orfluidic connectors.

In a multi-substrate device, according to some embodiments of theinvention, one or more of the substrates, such as the substrates 250-2,450-2 remain in a cartridge housing, while secondary substrates such asthe tiles 250-1, 450-1 are swapped for analyses of different samples.For example, samples may be loaded on several tiles, which are thenswapped in a cartridge for sequential analysis.

Multi-substrate devices have additional advantages. For example, thepacking of a column in a column tile and a column in a trap tile mayprogress more easily if the columns reside in different substratesrather than the same substrate.

Multi-substrate devices have other uses, in addition to those mentionedabove. For example, different substrates are optionally maintained atdifferent temperatures. For example, a temperature differential betweena trap column and a separation column may be more readily controlled ifthe columns reside in different substrates. In particular, this may bethe case where the substrates are formed of ceramic materials having arelatively high thermal conductivity. In some embodiments, activetemperature control is applied to one or more of the substrates. Forexample, one or more of the substrates can have heating and/or coolingfeatures, as described above.

Some preferred embodiments of the invention entail apparatus of reducedcost and size relative to existing apparatus, such as existinganalytical equipment based on LC-MS. Miniaturization provides manypotential benefits in addition to size reduction, for example: improvingreliability; reducing the quantity and cost of reagents, and the cost ofused-reagent disposal; and improve performance reducing dispersion inLC-related components. While preferred embodiments, described herein,relate to liquid chromatography, one of skill will recognize that theinvention may be applied to other separation techniques.

While the invention has been shown and described with reference tospecific preferred embodiments, it should be understood by those skilledin the art that various changes in form and detail may be made thereinwithout departing from the scope of the invention as defined by thefollowing claims. For example, multiple substrates are optionallyattached to one another in a more or less permanent manner, for example,using glue or some other bonding mechanism.

1. A method of analyzing proteins, comprising: providing a complexprotein-related sample comprising a plurality of components; loading asufficient quantity of the sample onto a trap column to overload thetrap column, wherein an outlet of the trap column is in fluidcommunication with an inlet of an analytical column; heating theanalytical column; heating the trap column to a greater temperature thanthe analytical column, thereby providing a temperature differentialbetween the trap and analytical columns; and pumping a solvent, to thetrap column, having a solvent composition profile that, in cooperationwith the temperature differential, causes at least some of thecomponents to elute sequentially from the trap column to the analyticalcolumn and focus on the analytical column prior to eluting from theanalytical column.
 2. The method of claim 1, wherein the trap column ispacked with particles having a diameter of greater than about 2 μm, andthe analytical column is packed with particles having a diameter of lessthan about 2 μm.
 3. The method of claim 1, wherein the particles of theanalytical column have a greater hydrophobicity than the particles ofthe trap column.
 4. The method of claim 1, wherein the trap column ismaintained at a temperature of greater than about 45° C. and theanalytical column is maintained at a temperature of less than about 45°C.
 5. The method of claim 1, wherein loading comprises maintaining thetrap column at a temperature of about 25° C. or less.
 6. The method ofclaim 1, wherein the analytical column has an inner cross-sectionaldimension in a range of about 50 μm to about 150 μm.
 7. An apparatus forchromatographic separation of a sample, comprising: a trap column; aseparation column in fluidic communication with the trap column; atrap-column heater; a separation-column heater; a solvent pump unit; anda control unit, including instructions, which, when implemented, causesthe apparatus to perform the steps of, loading a sufficient quantity ofa complex protein-related sample onto the trap column to overload thetrap column; heating the analytical column; heating the trap column,wherein the heated trap column has a greater temperature than the heatedanalytical column, thereby providing a temperature differential betweenthe trap and analytical columns; and pumping a solvent, to the heatedtrap column, having a solvent composition profile that, in cooperationwith the temperature differential, causes at least some of thecomponents to elute sequentially from the heated trap column to theheated analytical column and focus on the heated analytical column priorto eluting from the heated analytical column.
 8. The apparatus of claim7, wherein the trap column is packed with particles having a diameter ofgreater than about 2 μm, and the analytical column is packed withparticles having a diameter of less than about 2 μm.
 9. The apparatus ofclaim 8, wherein the particles of the analytical column have a greaterhydrophobicity than the particles of the trap column.
 10. The apparatusof claim 7, wherein the temperature of the heated trap column is greaterthan about 45° C. and the temperature of the heated analytical columnless than about 45° C.
 11. The apparatus of claim 7, wherein theanalytical column has an inner cross-sectional dimension in a range ofabout 50 μm to about 150 μm
 12. A method of chemical analysis,comprising: (a) providing a ceramic-particle-based and/or metal-basedmicrofluidic substrate defining a trap column and an analytical columnin fluidic communication with the trap column; (b) loading a sample onthe trap column while the trap column is at a temperature in a loadrange; (c) heating at least a portion of the substrate containing theanalytical column to provide a temperature above ambient during elutionof the sample through the analytical column, wherein the trap column isincidentally heated; (d) pumping a solvent to the trap column to elutethe sample components from the trap column to the analytical column atthe temperature above ambient, causing the components to elute from theanalytical column; (e) cooling at least a portion of the substratecontaining the trap column, after elution of the components from theanalytical column, to return the trap column to a temperature in theload range; and (f) repeating (b) through (e) for each of one or moresubsequent samples.
 13. The method of claim 12, wherein the analyticalcolumn has particles having a greater hydrophobicity than particles ofthe trap column.
 14. The method of claim 12, wherein the temperatureabove ambient of the heated analytical column about 35° C. or greater.15. The method of claim 12, wherein the analytical column has an innercross-sectional dimension in a range of about 50 μm to about 150 μm. 16.An apparatus for chromatographic separation of a sample, comprising: aceramic-particle-based and/or metal-based microfluidic substratedefining a trap column and an analytical column in fluidic communicationwith the trap column; a fluidic conduit having an outlet disposed todirect a fluid towards a location of the trap-column to cool at least aportion of the microfluidic substrate; a separation-column heating unitdisposed to heat at least a separation column portion of themicrofluidic substrate during separation of a sample; and a solvent pumpunit for pumping a solvent composition to an inlet of the trap column.17. The apparatus of claim 16, wherein an outlet of the trap column andan inlet of the analytical column are substantially co-located, in thesubstrate.
 18. The apparatus of claim 17, wherein a packing material ofthe trap column and a packing material of the analytical column arecontiguous.
 19. The apparatus of claim 16, wherein the analytical columnhas an inner cross-sectional dimension in a range of about 50 μm toabout 150 μm.
 20. A method of analyzing small molecules, comprising:cooling at least a portion of an analytical column proximate to an inletof the analytical column; loading, onto the cooled portion of theanalytical column, a nano-scale sample comprising a plurality ofdifferent small-molecule components; heating the analytical column topromote elution of the loaded components; and pumping a solvent, to theheated analytical column, to elute the components from the analyticalcolumn.
 21. The method of claim 20, wherein the analytical column has aninner cross-sectional dimension in a range of about 150 μm to about 500μm.
 22. The method of claim 21, wherein the analytical column isdisposed in a microfluidic substrate.